Strain-induced fetal type II epithelial cell differentiation is mediated via cAMP-PKA-dependent signaling pathway.

The signaling pathways by which mechanical forces modulate fetal lung development remain largely unknown. In the present study, we tested the hypothesis that strain-induced fetal type II cell differentiation is mediated via the cAMP signaling pathway. Freshly isolated E19 fetal type II epithelial cells were cultured on collagen-coated silastic membranes and exposed to mechanical strain for varying intervals, to simulate mechanical forces during lung development. Unstretched samples were used as controls. Mechanical strain activated heterotrimeric G-protein alpha(s) subunit, cAMP, and the transcription factor cAMP response element binding protein (CREB). Incubation of E19 cells with the PKA inhibitor H-89 significantly decreased strain-induced CREB phosphorylation. Moreover, adenylate cyclase 5 and CREB genes were also mechanically induced. In contrast, components of the PKA-independent (Epac) pathway, including Rap-1 or B-Raf, were not phosphorylated by strain. The addition of forskolin or dibutyryl cAMP to unstretched E19 monolayers markedly upregulated expression of the type II cell differentiation marker surfactant protein C, whereas the Epac agonist 8-pCPT-2'-O-Me-cAMP had no effect. Furthermore, incubation of E19 cells with the PKA inhibitor Rp-2'-O-monobutyryladenosine 3',5'-cyclic monophosphorothioate or transient transfection with plasmid DNA containing a PKA inhibitor expression vector significantly decreased strain-induced surfactant protein C mRNA expression. In conclusion, these studies indicate that the cAMP-PKA-dependent signaling pathway is activated by force in fetal type II cells and participates in strain-induced fetal type II cell differentiation.

Stretch activation of GTP-binding proteins in C2C12 myoblasts.

Mechanical stimulation has been proposed as a fundamental determinant of muscle physiology. The mechanotransduction of strain and strain rate in C2C12 myoblasts were investigated utilizing a radiolabeled GTP analogue to detect stretch-induced GTP-binding protein activation. Cyclic uniaxial strains of 10% and 20% at a strain rate of 20% s(-1) rapidly (within 1 min) activated a 25-kDa GTPase (183 +/- 17% and 186 +/- 19%, respectively), while 2% strain failed to elicit a response (109 +/- 11%) relative to controls. One, five, and sixty cycles of 10% strain elicited 187 +/- 20%, 183 +/- 17%, and 276 +/- 38% increases in activation. A single 10% stretch at 20% s(-1), but not 0.3% s(-1), resulted in activation. Insulin activated the same 25-kDa band in a dose-dependent manner. Western blot analysis revealed a panel of GTP-binding proteins in C2C12 myoblasts, and tentatively identified the 25-kDa GTPase as rab5. In separate experiments, a 40-kDa protein tentatively identified as Galpha(i) was activated (240 +/- 16%) by 10% strain at 1 Hz for 15 min. These results demonstrate the rapid activation of GTP-binding proteins by mechanical strain in myoblasts in both a strain magnitude- and strain rate-dependent manner.

Strain rate mechanotransduction in aligned human vascular smooth muscle cells.

Vascular smooth muscle cells (VSMCs) exist in a dynamic mechanical environment and can sense and respond to mechanical stimuli in vivo. Stretch is known to stimulate intracellular biochemical events, but the influence of the rate at which stretch is applied has not been extensively investigated. Also, most studies of VSMC mechanotransduction use cell culture models not aligned in the direction of stretch. We aligned human VSMC in the direction of uniaxial stretch to examine the importance of strain rate and cell orientation. We demonstrate strain rate profoundly affects stretch-induced phosphorylation of extracellular signal-regulated kinase (ERK)1/2. Low strain rate induced dephosphorylation while physiologic and high rates increased phosphorylation. Dephosphorylation at low strain rate was dependent on cell orientation matching the strain field. Pretreatment with GDPbetaS indicated G proteins are required for ERK1/2 phosphorylation at physiologic strain rate. Apyrase addition to scavenge extracellular ATP inhibited ERK1/2 regulation at low and physiologic strain rates. These results indicate strain rate and cell orientation are important components of mechanotransduction.

Rapid activation of Ras by fluid flow is mediated by Galpha(q) and Gbetagamma subunits of heterotrimeric G proteins in human endothelial cells.

OBJECTIVE: Temporal gradients in fluid shear stress have been shown to induce a proatherogenic phenotype in endothelial cells. The biomechanical mechanism(s) that enables the endothelium to respond to fluid shear stress requires rapid activation and signal transduction. The small G protein Ras has been identified as an early link between rapid mechanotransduction events and the effects of shear stress on downstream signal-transduction cascades. The aim of this study was to elucidate the upstream mechanotransduction signaling events mediating the rapid activation of Ras by fluid shear stress in human endothelial cells. METHODS AND RESULTS: Direct measurement of Ras-bound GTP and GDP showed that fluid-flow activation of Ras was rapid (10-fold within 5 seconds) and dose dependent on shear stress magnitude. Treatment with protein tyrosine kinase inhibitors or pertussis toxin did not significantly affect flow-induced Ras activation. However, activation was inhibited by transient transfection with antisense to Galpha(q) or the Gbetagamma scavenger beta-adrenergic receptor kinase carboxy terminus. Transfection with several Gbetagamma subunit isoforms revealed flow-induced Ras activation was most effectively enhanced by Gbeta1gamma2. CONCLUSIONS: These results suggest that the rapid, shear-induced activation of Ras is mediated by Galpha(q) through the activity of Gbetagamma subunits in human vascular endothelial cells.

Strain and strain rate activation of G proteins in human endothelial cells.

The endothelium is known to sense and respond to its physical environment, but the underlying mechanisms and early events of endothelial cell mechanotransduction are not well understood. The present study measured G protein activation by mechanical strain in human umbilical vein endothelial cells (HUVEC) directly by photoincorporation of a hydrolysis resistant, radiolabeled GTP analog. Ten percent uniaxial strain at a strain rate of 20% s(-1) over 1min activated a 38kDa Galpha subunit 167+/-17% relative to controls, while 2% cyclic strain failed to significantly activate the protein (117+/-19%). A single cycle of 10% strain at 20% s(-1) strain rate activated the Galpha subunit 152+/-25%, while activation at the same strain but lower strain rate (0.3% s(-1)) was not significantly different from controls (116+/-12%). Western blot analysis identified the 38kDa protein as Galpha(q/11). These results demonstrate the rapid activation of G proteins in HUVEC by cyclic uniaxial strain in a strain- and strain rate-dependent manner.